High mobility group protein (HMGB1) is a non-histone DNA binding protein that facilitates transcription. Recently, investigators have shown that HMGB1 has other roles that may be critical in the development of sepsis and septic shock. HMGB1 is released as a late mediator of sepsis (i.e., after 8 to 15 hours) from mononuclear cells stimulated with TNF, IL-1, or endotoxin. It is detected in the blood of septic mice and in septic patients and it worsens outcome when given to septic mice. It plays a role in migrating axons of neurons in the developing brain and it activates plasminogen. Some of the actions are through the RAGE receptor (receptor for advanced glycation products) which plays a role in chronic inflammation in diabetes. This novel axis of inflammation remains to be characterized in human sepsis. In order to study the target cells and contribution of HMGB1 to acute human inflammation, we have produced recombinant human HMGB1 in bacterial and yeast expression systems and have induced the release of HMGB1 from primary human cell lines. This has afforded us the opportunity to study native and recombinant protein effects in target cells including endothelium (Blood 2003;101:2652), respiratory epithelium, and mononuclear cells.[unreadable] [unreadable] In addition, we are developing biologically active peptide fragments of the intact molecule in order to study structure function relationships. Cell lines and migrating human cells from humans challenged with endotoxin will be studied for the expression of RAGE and their responses to HMGB1. HMGB1 will also be studied in blood and inflammatory lavage obtained from volunteers challenged with endotoxin (protocol 92-CC-0141). Oligonucleotide gene arrays will be used to study the inflammatory axis initiated by HMGB1 on target cells. These data should provide important new information regarding the role of HMGB1 in acute human inflammation to bacterial products.